Small interfering RNAs (siRNAs) are commonly employed, both individually and on a genome-wide scale, to degrade specific mRNAs and thus allow testing of the cellular functionality for the proteins encoded by the mRNA. siRNA depletion experiments can be extended further using “rescue” procedures in which the target protein is re-expressed from a transiently transfected vector that encodes an altered mRNA that is resistant to siRNA silencing. Such rescue experiments can be useful for confirming siRNA specificity because the exogenously expressed protein should rescue the loss-of-function phenotype. The experiment can also enable genetic analysis in cultured cells because the functional effects of specific mutations can be tested.
Phenotypic rescue experiments can fail, however, when the rescuing protein is expressed at, for example, such a high level that it dominantly inhibits the pathway of interest. This problem can often be alleviated by reducing the quantity of transfected expression vector, but this approach can fail if the overall transfection efficiency is reduced.